Summary Triple negative breast cancer (TNBC), one of the most aggressive types of cancer that manifests resistance to virtually all conventional targeted therapies and hormonal treatments (e.g. tamoxifen, TAM), is particularly en- riched in a high content of cancer stem cells (CSCs), a sub-population of cancer cells account for cancer pro- gression, therapy resistance, and recurrence. In recent years, a widely prescribed diabetes drug, Metformin (metformin hydrochloride), has shown promise in treatment of breast cancer, including TNBC. However, the molecular target(s) and the key mechanism underlying metformin mediated anti-tumor effects, particularly for the regulation of CSCs, still remain elusive. Our preliminary data for this study provides novel evidence showing that metformin treatment in TNBC cells leads to activation of selective microRNAs (MI-miRs: miR-200c, miR-152, miR-448, miR-377) that to- gether target and suppress PKC?, a cell polarity protein that plays a central role in maintenance of the self-re- newing stem cell fate decision. Inhibition of PKC? by metformin in turn directs luminal differentiation of the TNBC-CSCs with re-expression of ER?, which allows for sensitization of TNBC cells to TAM in vitro. Further- more, the pilot findings also reveal that a metformin-resistant CSC population would emerge under long term metformin treatment and exhibit significantly enhanced expression of histone methyltransferase EZH2, which potentially mediates silencing of the MI-miRs to sustain PKC? expression and thereby promotes the resistant CSC pool and tumor progression. Based on the evidence, it is our expectation that a precise delineation of the underlying mechanism governing CSC response (self-renewal vs. differentiation) to metformin likely holds the key to the development of an effective and stratified therapeutic strategy that can be used to eradicate TNBC. To test the hypothesis, we propose to first determine the mechanism(s) governing metformin-induced CSC differentiation and TAM sensitization (Aim 1) by delineating PKC?-mediated signaling regulation underlying TNBC response to metformin-induced luminal differentiation and TAM sensitization. Next, we will determine strategies to overcome metformin resistance and enhance the therapeutic effect of metformin on TNBC (Aim 2) by testing whether metformin combined with TAM will be effective in abrogation of TNBC stemness as well as tumor growth in TNBC patient-derived xenograft mouse models. We will also examine whether a clinical trial tested selective EZH2 inhibitor, tazemetostat, can re-direct metformin-resistant CSCs to metformin-induced differentiation and re-sensitize resistant TNBC to TAM in vivo. Furthermore, using a newly-established TNBC animal model (Brca1f/fp53+/-;Ezh2) and the annotated patient specimens from a phase III metformin trial in breast cancer, we will determine the role of metformin-induced signaling cascade in TNBC development and treatment response (Aim 3), leading to novel stratification biomarkers with clinicopathological and prognostic relevance to ensure effective metformin/TAM treatment regimens.